Quantitative assessment of prion infectivity in tissues and body fluids by real-time quaking-induced conversion

Abstract

Prions are amyloid-forming proteins that cause transmissible spongiform encephalopathies through a process involving the templated conversion of the normal cellular prion protein (PrP(C)) to a pathogenic misfolded conformation. Templated conversion has been modelled in several in vitro assays, including serial protein misfolding amplification, amyloid seeding and real-time quaking-induced conversion (RT-QuIC). As RT-QuIC measures formation of amyloid fibrils in real-time, it can be used to estimate the rate of seeded conversion. Here, we used samples from deer infected with chronic wasting disease (CWD) in RT-QuIC to show that serial dilution of prion seed was linearly related to the rate of amyloid formation over a range of 10(-3) to 10(-8) µg. We then used an amyloid formation rate standard curve derived from a bioassayed reference sample (CWD+ brain homogenate) to estimate the prion seed concentration and infectivity in tissues, body fluids and excreta. Using these methods, we estimated that urine and saliva from CWD-infected deer both contained 1-5 LD50 per 10 ml. Thus, over the 1-2 year course of an infection, a substantial environmental reservoir of CWD prion contamination accumulates.

Fig. 1.

Quantitative analysis of prion amyloid seeding levels with the RT-QuIC assay. (a) Model of the seeded RT-QuIC assay. Amyloid amplification is not observed until ThT fluorescence levels reach a detectable level. We estimate the amyloid formation rate by using the time at which a RT-QuIC assay crosses a defined threshold, termed C_t. When C_t values or the inverse of C_t values are plotted on a log scale, a linear relationship should be observed (Gibson et al., 1996; Murphy et al., 1990). (b) Reactions in the RT-QuIC assay were determined to be positive when a threshold of 5 sd above the initial fluorescence is reached. (c) Analysis of a CWD+ brain pool from 10⁻¹ to 10⁻⁸ dilution of a 10 % homogenate. The mean±sd amyloid formation rate is displayed for each dilution. Each point is derived from eight replicates from three experiments. No reaction is observed at 10⁻¹ or 10⁻² and a slower reaction rate is seen at 10⁻³ dilution; 10⁻⁴ to 10⁻⁷ show a linear response as predicted.

Fig. 2.

PTA precipitation of CWD+ brain homogenate dilutions to enhance detection. The amyloid formation rate with or without PTA precipitation is shown for 10⁻¹ to 10⁻⁴ dilutions of a 10 % CWD+ brain pool homogenate. No reaction is seen for 10⁻¹ or 10⁻² in non-PTA-precipitated samples. A 10⁻² dilution from PTA-precipitated samples shows a similar amyloid formation rate to a 10⁻³ dilution of non-PTA-treated CWD+ brain. PTA precipitation does not substantially affect the amyloid formation rate of more dilute samples. Each result represents the mean±sd and is derived from four replicates from two experiments.

Fig. 3.

PrP^Res levels detected by western blotting correlate with quantitative RT-QuIC assessment of the same sample. (a) Lanes 1 and 2, western blot analysis of CWD– brain sample with and without proteinase K (PK) treatment. Lanes 3 and 4, proteinase K-resistant PrP^Res samples from the obex and mid-brain of deer 776 that had advanced terminal disease. Lanes 5 and 6, corresponding samples from deer 783 that did not have robust accumulation of PrP^Res in any brain sample analysed. Five times the brain equivalents of lane 3 and 4 were added to lanes 5 and 6 so a signal could be observed. (b) Quantitative RT-QuIC analysis of the same brain samples shown in the western blot in (a). Both samples from deer 776 that had a robust western blot signal (lanes 3 and 4 above) had significant amyloid seeding capacity across more than five orders of magnitude. Both samples from deer 783 had less amyloid amplifying activity compared with 776. Each point represents the mean±sd and is derived from four replicates from two experiments.

Fig. 4.

Quantification of a CWD+ brain sample after the end-point dilution bioassay. (a) Percentage of cervidized transgenic mice that succumbed to CWD at each dilution point. Drop-lines (dotted) denote the LD₅₀. LD₅₀ is calculated to be 13.8 pg. (b) RT-QuIC quantitative analysis of the same sample used for the end-point dilution bioassay in (a). The solid line is the best-fit semi-log linear regression of the bioassayed brain sample analysed by RT-QuIC. Dashed lines are the 95 % confidence intervals of the best-fit curve. The vertical dotted line is the LD₅₀ (µg CWD+ brain). The horizontal dotted line is the calculated amyloid formation rate (h^–1) for 1 LD₅₀. Each point for RT-QuIC represents the mean±sd and is derived from six replicates from three experiments.

Fig. 5.

Analysis and quantification of prion seeding activity in various tissues by RT-QuIC. (a) Amyloid formation rate determination over multiple log dilutions of left ventricle, jejunum, pancreas and spleen from a terminal CWD+ white-tailed deer. All tissues were harvested at terminal disease. The dotted line, which is the same in each panel, represents the rate values of the obex from the same animal. Each point represents the mean±sd and is derived from four replicates from two experiments. (b) LD₅₀ equivalents (mean±sd) in 0.2 ng of obex, left ventricle, pancreas, jejunum and spleen.

Fig. 6.

Analysis and quantification of prion seeding activity in saliva and urine using RT-QuIC. (a) Amyloid formation rates from the obex, saliva and urine from the same animal. (Top) Two time points post-infection for saliva and urine (6 and 9 months for saliva; 6 and 22 months for urine) from deer 773 are shown. (Middle) Obex, saliva and urine from deer 812. (Bottom) Obex, saliva and urine from deer 817. Each dot represents one replicate. Each sample was analysed in at least two experiments. Bar, sd; centre line, mean. (b) Estimation of LD₅₀ equivalents (mean±sd) for 1.0 ml saliva and urine for the same samples in (a).